One focus of the group is to study how organisms sense environmental signals and transduce the signals into changes in gene expression and cell physiology. Specifically, we are examining the E. coli and S. cerevisiae responses to oxidative stress. Reactive oxygen species can lead to the damage of almost all cell components (DNA, lipid membranes, and proteins) and have been implicated as causative agents in several degenerative diseases. Most organisms have an adaptive response to defend against oxidants. For example, treatment of both bacterial and yeast cells with low doses of hydrogen peroxide results in the induction of a distinct group of proteins, the decreased expression of other proteins, and resistance to killing by subsequent higher doses of hydrogen peroxide. In bacterial cells, the key regulator of the inducible defenses against hydrogen peroxide is the OxyR transcription factor. We discovered that OxyR is both the sensor and transducer of the oxidative stress signal; the oxidized but not the reduced form of the purified regulator can activate transcription in vitro. OxyR is activated by the formation of an intramolecular disulfide bond between C199 and C208 and is deactivated by enzymatic reduction by glutaredoxin 1 together with glutathione. Structural studies showed that formation of the C199-C208 disulfide bond leads to a large conformational change. We now are examining the chemical basis of OxyR sensitivity to hydrogen peroxide and the roles of all of OxyR target genes. Compared to the bacterial response to hydrogen peroxide, relatively little is known about the cellular mechanisms used by higher cells to sense and protect against oxidative damage. To initiate studies of the oxidative stress response in eukaryotes, we constructed isogenic Saccharomyces cerevisiae strains carrying mutations in known signal transduction pathways and compared the oxidant sensitivities and whole genome expression patterns of these mutants. These studies confirmed that the Yap1 transcription factor is critical for resistance to hydrogen peroxide. Thus we initiated studies to purify and characterize the Yap1 protein. We also are carrying out genetic screens for yeast mutants with altered sensitivities to hydrogen peroxide in order to identify new components of the eukaryotic response to oxidative stress. A second focus of this group is to elucidate the functions of small, untranslated, regulatory RNAs. One of the OxyR-induced genes encodes the OxyS RNA, which acts as a pleiotropic regulator and as an antimutator. OxyS RNA action is dependent on the Sm-like Hfq protein, and biochemical experiments showed that Hfq acts as a chaperone to facilitate OxyS RNA basepairing with its target mRNAs. We also found that another abundant, small E. coli RNA, 6S RNA, binds and modifies RNA polymerase. Recently, comparative genomics and microarrays were used to identify novel small RNAs in E. coli. These approaches led to the identification of 17 new small RNAs whose functions we are characterizing.